Bradykinin and ATP stimulate L-arginine uptake and nitric oxide release in vascular endothelial cells.

The effects of bradykinin and ATP on L-arginine transport and nitric oxide (NO) production were studied in porcine aortic endothelial cells cultured and perfused on microcarriers and deprived of L-arginine for 24 h. Stimulation of cells with bradykinin (100 nM) or ATP (100 microM) resulted in a rapid increase in L-arginine uptake and NO release. In the presence of nitro-L-arginine (100 microM), an inhibitor of NO synthase, the stimulatory effect of bradykinin on L-arginine uptake was partially inhibited while NO release was completely abolished. Nitro-L-arginine alone was not an inhibitor of basal L-arginine transport, suggesting that its inhibitory action was not directly on the L-arginine transporter but a result of the inhibition of NO generation. These data indicate that during agonist-stimulated NO production there is a concomitant increase in the transport of L-arginine into endothelial cells providing a mechanism for the continual generation of NO.     

Fanconi anemia: evidence for linkage heterogeneity on chromosome 20q

Fanconi anemia is a rare autosomal recessive disorder in which affected individuals are predisposed to acute myelogenous leukemia and other malignancies. We report the results of a genetic linkage study involving 34 families enrolled in the International Fanconi Anemia Registry. A significant lod score was obtained between D20S20, an anonymous DNA segment from chromosome 20q, and Fanconi anemia (Zmax 3.04, theta max = 0.12). However, six other anonymous DNA segments from chromosome 20q, including D20S19, which is highly polymorphic and tightly linked to D20S20, showed no or only weak evidence for linkage to Fanconi anemia. An admixture test revealed significant evidence for linkage heterogeneity (chi 2 = 6.10, P = 0.01) at the D20S19 locus. Lod scores suggestive of linkage between Fanconi anemia and this locus were obtained with two of the largest kindreds studied (lods = 2.6 and 2.1, at theta = 0.001). Thus, our data support the provisional assignment of a Fanconi anemia gene to chromosome 20q.     

New mechanical aid to physiotherapy in cystic fibrosis.

The force of impact and frequency of percussion by physiotherapists and parents of children with cystic fibrosis were analysed on a special test rig and incorporated in a prototype percussor. In adult volunteers and cadavers a maximum intrathoracic pressure could be achieved by a critical frequency of mechanical percussion which was higher than that reached by physiotherapists and parents. Consequently the percussor was redesigned to operate at this optimum frequency. It was then discovered that if the percussor was pressed firmly enough against the chest, this maximum intrathoracic pressure could be indicated by quivering of the voice. In a continuing study of intrathoracic pressures obtained mechanically and manually the Salford percussor''s produced higher pressures than the physiotherapists'' and maintained them constantly, while the physiotherapists'' efforts and results varied from one to another. Hospital and domiciliary use of the percussor have shown it to help in the first stage of the physiotherapy routine for patients with cystic fibrosis. The percussor should enable adolescents and adults to treat themselves and encourage twice-daily and more effective chest treatments. It is easy to apply and its speed and efficiency should enable parents to improve the quality of their chest therapy for younger children at home. Its long-term benefits are difficult to assess because of the nature of the disease.     

Moths complement bumblebee pollination of red clover: a case for day-and-night insect surveillance

Recent decades have seen a surge in awareness about insect pollinator declines. Social bees receive the most attention, but most flower-visiting species are lesser known, non-bee insects. Nocturnal flower visitors, e.g. moths, are especially difficult to observe and largely ignored in pollination studies. Clearly, achieving balanced monitoring of all pollinator taxa represents a major scientific challenge. Here, we use time-lapse cameras for season-wide, day-and-night pollinator surveillance of Trifolium pratense (L.; red clover) in an alpine grassland. We reveal the first evidence to suggest that moths, mainly Noctua pronuba (L.; large yellow underwing), pollinate this important wildflower and forage crop, providing 34% of visits (bumblebees: 61%). This is a remarkable finding; moths have received no recognition throughout a century of T. pratense pollinator research. We conclude that despite a non-negligible frequency and duration of nocturnal flower visits, nocturnal pollinators of T. pratense have been systematically overlooked. We further show how the relationship between visitation and seed set may only become clear after accounting for moth visits. As such, population trends in moths, as well as bees, could profoundly affect T. pratense seed yield. Ultimately, camera surveillance gives fair representation to non-bee pollinators and lays a foundation for automated monitoring of species interactions in future.     

Developability studies before initiation of process development

Monoclonal antibodies constitute a robust class of therapeutic proteins. Their stability, resistance to stress conditions and high solubility have allowed the successful development and commercialization of over 40 antibody-based drugs. Although mAbs enjoy a relatively high probability of success compared with other therapeutic proteins, examples of projects that are suspended due to the instability of the molecule are not uncommon. Developability assessment studies have therefore been devised to identify early during process development problems associated with stability, solubility that is insufficient to meet expected dosing or sensitivity to stress. This set of experiments includes short-term stability studies at 2−8 þC, 25 þC and 40 þC, freeze-thaw studies, limited forced degradation studies and determination of the viscosity of high concentration samples. We present here three case studies reflecting three typical outcomes: (1) no major or unexpected degradation is found and the study results are used to inform early identification of degradation pathways and potential critical quality attributes within the Quality by Design framework defined by US Food and Drug Administration guidance documents; (2) identification of specific degradation pathway(s) that do not affect potency of the molecule, with subsequent definition of proper process control and formulation strategies; and (3) identification of degradation that affects potency, resulting in program termination and reallocation of resources.     

Functional integrity of intravenous immunoglobulin following irradiation with a virucidal dose of gamma radiation.

Although intravenous immunoglobulins (IVIG) and other plasma therapeutics have had a relatively good safety record, improved methods for viral clearance are constantly being evaluated and incorporated into new manufacturing processes. Gamma irradiation has been used routinely to assure sterility of healthcare products and medical devices, but it has not been applied successfully as a viral inactivation method for biologics. We examine whether virucidal doses of gamma irradiation (50 kGy) can be delivered to a manufacturing intermediate form of IVIG, a fractionated plasma paste, with negligible effect on structural and functional integrity of purified IgG product. Immunoglobulins from paste were examined for radiation-induced damage by SDS-PAGE and ELISAs utilizing viral antigens specific for rubella, CMV and mumps. Fc domain integrity was assessed by immunoblotting, quantitatively comparing the binding of irradiated and non-irradiated materials to cell surface Fcgamma receptors, and by employing quantitative RT-PCR to study the kinetics of accumulation of mRNA for the immune modulatory cytokines IL-1alpha, IL-1beta, IL-4, IL-8, IFNgamma, and TNFalpha. The results demonstrate that Fab and Fc domains of IVIG remain essentially intact and functional after gamma irradiation to virucidal doses, suggesting that this method could be used to enhance the safety of IVIG products.     

An ITS-based phylogenetic framework for the genus Vorticella: finding the molecular and morphological gaps in a taxonomically difficult group

Vorticella includes more than 100 currently recognized species and represents one of the most taxonomically challenging genera of ciliates. Molecular phylogenetic analysis of Vorticella has been performed so far with only sequences coding for small subunit ribosomal RNA (SSU rRNA); only a few of its species have been investigated using other genetic markers owing to a lack of similar sequences for comparison. Consequently, phylogenetic relationships within the genus remain unclear, and molecular discrimination between morphospecies is often difficult because most regions of the SSU rRNA gene are too highly conserved to be helpful. In this paper, we move molecular systematics for this group of ciliates to the infrageneric level by sequencing additional molecular markers—fast-evolving internal transcribed spacer (ITS) regions—in a broad sample of 66 individual samples of 28 morphospecies of Vorticella collected from Asia, North America and Europe. Our phylogenies all featured two strongly supported, highly divergent, paraphyletic clades (I, II) comprising the morphologically defined genus Vorticella. Three major lineages made up clade I, with a relatively well-resolved branching order in each one. The marked divergence of clade II from clade I confirms that the former should be recognized as a separate taxonomic unit as indicated by SSU rRNA phylogenies. We made the first attempt to elucidate relationships between species in clade II using both morphological and multi-gene approaches, and our data supported a close relationship between some morphospecies of Vorticella and Opisthonecta, indicating that relationships between species in the clade are far more complex than would be expected from their morphology. Different patterns of helix III of ITS2 secondary structure were clearly specific to clades and subclades of Vorticella and, therefore, may prove useful for resolving phylogenetic relationships in other groups of ciliates.     

Novel polymorphic microsatellite loci for the protogynous hermaphrodite slinger sea bream (Chrysoblephus puniceus, Sparidae)

As a result of the global decline of fish stocks, an increasing number of fish species are becoming targets of heavy exploitation, often concomitantly with a lack of biological knowledge on their structure and demographics. Here we present 11 new polymorphic microsatellite loci, isolated from the slinger sea bream (Chrysoblephus puniceus, Sparidae), a relatively recent target of coastal fisheries in eastern South Africa. Levels of genetic diversity were assessed in 39 individuals collected from the KwaZulu-Natal coast (Park Rynie, South Africa). Observed and expected heterozygosities varied between 0.39 and 0.97 and between 0.53 and 0.96, respectively. One locus (SL35) showed significant heterozygote deficiency and linkage disequilibrium was detected between SL35 and SL1. Importantly, five of these microsatellites cross-amplify in Cheimerius nufar, a sympatric species also subjected to exploitation.     

Early Social Networks Predict Survival in Wild Bottlenose Dolphins

A fundamental question concerning group-living species is what factors influence the evolution of sociality. Although several studies link adult social bonds to fitness, social patterns and relationships are often formed early in life and are also likely to have fitness consequences, particularly in species with lengthy developmental periods, extensive social learning, and early social bond-formation. In a longitudinal study of bottlenose dolphins (Tursiops sp.), calf social network structure, specifically the metric eigenvector centrality, predicted juvenile survival in males. Additionally, male calves that died post-weaning had stronger ties to juvenile males than surviving male calves, suggesting that juvenile males impose fitness costs on their younger counterparts. Our study indicates that selection is acting on social traits early in life and highlights the need to examine the costs and benefits of social bonds during formative life history stages.